Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, WEEV respectively) are arthropod-borne alphaviruses that cause periodic epizootics in the Americas. No licensed vaccines exist for these pathogens, and the experimental vaccines used under Investigational New Drug status suffer from high reactogenicity or poor immunogenicity. Vaccines against specific viruses causing encephalitis, including EEEV and WEEV, are regarded as High Priority Biodefense Products by the NIAID. VEEV has been "weaponized" for use in biowarfare, due largely to its considerable stability and high infectivity in aerosols. EEEV and WEEV are less well studied than VEEV, but both have potential uses as agents of biowarfare or bioterrorism. EEEV is in fact the most lethal of arboviruses. Clinical data of naturally acquired infection by WEEV suggest that the disease is less severe. However, by analogy to VEEV, aerosol exposure to EEEV and WEEV could be even more lethal. The present Proposal to develop improved immunogens for vaccines against EEEV and WEEV involves a collaboration between Maxygen and the USAMRIID laboratory of Dr. Connie Schmaljohn. Substantial improvements in the immunogenicity of the envelope glycoproteins of VEEV have already been obtained using in vitro multigene DNA recombination and directed molecular evolution. This demonstrates the potential of this approach for improving EEEV and WEEV immunogens. In this Phase I SBIR submission, we will focus on improving the immunogenicity of envelope glycoproteins from each of EEEV and WEEV separately. A separate Phase II SBIR application will target the creation of a single multivalent vaccine for VEEV, EEEV, and WEEV. The Specific Aims of the current Phase I SBIR submission are: (1) create expression libraries of recombined genes producing variants of the envelope glycoproteins from EEEV and WEEV; (2) perform prescreening of individual clones from the expression libraries; and (3) evaluate the immunogenicity of chimeric clones in mice using DNA vaccines. [unreadable] [unreadable]